Purification and characterization of human serum galactosyltransferase (lactose synthetase A protein).
A galactosyltransferase, which transfers galactose from UDP-galactose to N-acetylglucosamine, was purified 286,000-fold to homogeneity with 40% yield from human plasma by repeated affinity chromatography on alpha-lactalbumin-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with molecular weight of 49,000. The enzyme is a glycoprotein with 11% by weight carbohydrate, which seems to have only asparagine-N-acetylglucosamine linkage-type carbohydrate chains. The enzyme showed characteristic changes in activity at different alpha-lactalbumin concentrations, indicating that the enzyme is the A protein of lactose synthetase. Km values for the substrates were found to be 0.056 mM for UDP-galactose, 3.2 mM for GlcNAc, and 0.44 mM for Mn2+, and in the presence of alpha-lactalbumin, 3.4 mM for Glc, and 0.20 mM for Mn2+. The activity of the enzyme was neutralized by anti-enzyme antibody, but the antibody did not neutralize the bovine milk galactosyltransferase (A protein) activity.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000494:pharmacology; D007788:Lactose Synthase / Q000097:blood* / Q000302:isolation & purification; D009237:N-Acetyllactosamine Synthase; D013379:Substrate Specificity
D000596:Amino Acids; D002241:Carbohydrates; D007768:Lactalbumin; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase